人抑制素不同亚基抗原表位表达及抗体制备
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江苏省社会发展基金资助(BS2007014)


Expression of epitopes of human inhibin subunits and preparation of its antibodies
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    摘要:

    目的:根据人抑制素α-βA-βB 3个亚基特点,选择各亚基特定的抗原表位进行融合表达,并制备相应的多克隆抗体-方法:根据各亚基抗原表位氨基酸序列,合成其基因序列,并选用大肠杆菌偏爱密码子,插入pGEX-4T-2中作融合表达,纯化表达产物,免疫绵羊,制备抗体,测定了抗体水平-结果:经酶切鉴定,分别获得抑制素α-βA-βB与pGEX-4T-2连接的阳性重组子pGEX-INHA-pGEX-INHBA-pGEX-INHBB-其表达产物GST-INHA-GST-INHBA-GST-INHBB及纯化后融合蛋白,经考马斯亮蓝染色鉴定,大小正确-用常规免疫法免疫绵羊抗体,经ELISA检测抗体效价,结果显示,直接提取融合蛋白GST-INHA-GST-INHBA-GST-INHBB免疫绵羊抗体效价分别为:1∶64-1∶128-1∶32,纯化后的融合蛋白GST-INHA-GST-INHBA-GST-INHBB免疫绵羊抗体效价分别为:1∶32-1∶32-1∶64-结论:可在本研究所制备的抗体的基础上建立测定血清抑制素水平的免疫诊断方法-

    Abstract:

    Objective:To select epitopes of human inhibin subunits for fusion expression and antibody preparation according to the features of α,βA and βB. Methods:Based on amino acids sequences of inhibin subunits α(1-32),βA(82-114) and βB(101-134),the genes fragments were synthesized using preferred codons,which was inserted into pGEX-4T-2,and expressed in E. coli. The protein was purified for immunization of sheep antibody preparation. The titer of antibodies were determined by ELISA. Results:The positive clones named pGEX-INHA,pGEX-INHBA,pGEX-INHBB were choiced using enzyme digestion. The products of dones:GST-INHA,GST-INHBA,GST-INHBB,and the purified protein were identified with Commassie Blue Staining,then used to sheep immunization. The results showed that the titer of antibodies were 1∶64,1∶128 and 1∶32 using GST-INHA,GST-INHBA,and GST-INHBB,respectively,and 1∶32,1∶32 and 1∶64 using purified GST-INHA,GST-INHBA,and GST-INHBB,respectively. Conclusion:These antibodies can be used as diagnostic reagent of enzyme immunoassay detecting serum inhibin level.

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陈〓华,王〓玢,王俊霞,陈〓骞.人抑制素不同亚基抗原表位表达及抗体制备[J].南京医科大学学报(自然科学版),2009,29(12):1703-

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  • 收稿日期:2009-06-20
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